Study on small ruminant brucellosis and owners awareness in two selected districts of southern region, Ethiopia

Abstract Introduction Brucellosis is one of the infectious diseases that has the greatest impact on the productivity of sheep and goats. A cross‐sectional study followed by a simple random sampling technique was used to investigate the seroprevalence of brucellosis (Rose Bengal plate test; RBPT and complement fixation test; CFT) in small ruminants and its related risk variables from November 2019 to June 2020 in Kolme and Abala Abaya districts. A questionnaire was also given to owners to assess their existing knowledge of the disease. Result Using the RBPT and CFT, 28 (4.1%) and 23 (3.33%) of the 690 animals were found to be seropositive for brucellosis, respectively. In this study, the seroprevalence of brucellosis detected in the Kolme district (5.3%) was greater when compared to Abala Abaya (1.0%). The odds of Brucella infection were greater for goats (odds ratio [OR] 6.0, 95% confidence interval [CI] 16 0.8–44.9) than for sheep. The odds of adult animals (OR 0.05, 95% CI 0.03–0.07) being positive for brucellosis was higher than young animals. A statistically significant difference in the seropositivity of brucellosis was detected in univariate logistic regression among districts, different age groups, herd size, parity number, and reproductive health problems except for species and sex, but in multivariate logistic regression, only reproductive health problems were revealed a statistically significant difference. Out of 138 families, 100% of respondents were unaware of brucellosis, 94.5% drink raw milk, and 74% handle animals with retained fetal membranes with their bare hands. Conclusion This study showed that brucellosis was a widely spread disease in the study areas and poses a substantial public health danger. To reduce the spread of the disease in small ruminants, public health risks, and economic losses, stringent vaccination application and awareness of personal hygiene are critical.


INTRODUCTION
Goats and sheep are major domestic animals in African tropical livestock production systems, accounting for 21% of the global small ruminant population (Ashenafi et al., 2007). Ethiopia is one of East Africa's developing countries, with 31.30 million sheep and 32.74 million goats primarily raised in the country's lowland and pastoral regions (Addis & Desalegn, 2018). Because of their high proliferation rate and great ability to adapt to various environmental conditions, these small ruminants represent an important export commodity that significantly contributes to the livelihood and national economy of rural farmers as a source of food (milk and meat), wool, skins, source of income, and monetary asset, especially in pastoral and lowland areas of Ethiopia (Adams et al., 2021). Small ruminants are a vast resource; however, production from this valuable asset is not fully realized due to several technical and non-technical factors, including nutritional deficiency, husbandry difficulties, water scarcity, inadequate marketing, and losses associated with infectious diseases, primarily brucellosis (Ashenafi et al., 2007;Geletu et al., 2021;Tewodros & Dawit, 2015).
Brucellosis is a zoonotic disease (Franc et al., 2018) that is caused by microscopic coccobacilli bacteria of the genus Brucella. This Gramnegative bacterium is slow growing and capable of surviving and replicating within epithelial cells, placental trophoblasts, dendritic cells, and macrophages (Tekle et al., 2019).
In goats and sheep, Brucella melitensis produces the disease; however, in cattle, Brucella abortus can cause clinical brucellosis, and in rams, Brucella ovis causes epididymitis (Kusiluka & Kambarage, 1996). The disease affects the animal's reproductive system, causing significant production and productivity losses such as decreased milk production, abortion, stillbirth, acute orchitis, weak offspring, weight loss, culling, and condemnation of infected animals due to infertility, lameness, and trade and export restrictions (Makita et al., 2011).
In endemic areas, brucellosis is primarily transmitted between animals through direct contact with infected animals or contact with an environment that has been contaminated with infected birthing tissues or fluids, including the placenta, aborted fetuses, or uterine discharge.
Humans most frequently acquire brucellosis by consuming infected raw animal products (raw, unpasteurized milk, and soft cheeses) but can also develop the disease following contact with infected animal tissues or secretions (especially unpasteurized milk and soft cheeses) (Gupte & Kaur, 2015;Radostits et al., 2006). Different species of Brucella and their biovars has distinct distributions. The bacterium B. abortus can be found all over the world.
Brucella melitensis and Brucella suis have a sporadic distribution (Acha & Szyfres, 2003). The disease's occurrence in animals is related to a variety of factors. Animals' age, sex, and reproductive status are all host variables that can influence their susceptibility to infection (Nicoletti, 2010). Farmers, veterinarians, and others in the livestock industry are at high risk of contracting the disease (Sintayehu et al., 2015;Tewodros & Dawit, 2015).
The World Health Organization has designated brucellosis as one of the world's main 'neglected zoonotic diseases' , owing to the disease's disproportionate impact on low-income countries (Franc et al., 2018). In Ethiopia, the first case of brucellosis was reported almost 50 years ago (Domenech & Lefevre, 1974). Many studies detailing the prevalence and associated risk factors of small ruminant brucellosis throughout different parts of Ethiopia have been reported (Ashagrie et al., 2011;Asmare et al., 2013;Ferede et al., 2011;Melese, 2016;Sintayehu et al., 2015;Teshale et al., 2006;Tewodros & Dawit, 2015;Yesufa et al., 2011). Ethiopia is a large country with a huge livestock population, and previously published work has only described seroprevalence for specific areas and not the whole country (Tamrat et al., 2020). Furthermore, no published research work on small ruminant brucellosis was found in the current study region. Therefore, this study was initiated to investigate the seroprevalence and the associated risk factors of small ruminant brucellosis in the Kolme and Abala Abaya districts.

Study area
The study was undertaken in the Southern Nation Nationalities Peo-

Study populations
This study focused on indigenous sheep and goats in the Kolme and Abala Abaya districts, which were kept in the extensive production system. The study comprised sheep and goats aged 6 months and above, as well as both sexes in the selected flock that had never been vaccinated against brucellosis. Information regarding putative risk factors including origins of the animal, species, sex category, parity, and reproductive F I G U R E 1 Map of the Kolme and Abala Abaya districts health concerns (abortion, stillbirth, retained fetal membrane) was documented as previously described (Abebe & Yami, 2008;Shively, 1985).
The age of sheep and goats was determined based on their dental eruption patterns. Animals were classified as young (1-3 years old) if they had four or fewer permanent teeth and adult (4-5 years old) if they had more than four permanent teeth (Steel, 1996;Vatta et al., 2006).
From December 2019 to May 2020, a cross-sectional study was carried out in the Kolme and Abala Abaya districts to investigate the seroprevalence and the associated risk factors of small ruminant brucellosis.

Sample size determination
The sample size was calculated according to the approach suggested by Thrusfield (2018). The seroprevalence of small ruminant brucellosis was estimated at 5.9% using previously reported results in districts near those studied herein (Melese, 2016;Wakene et al. 2017). Six hundred and ninety goats and sheep were sampled in this study, which is twice as large as the sample size calculated to achieve a level of significance of 0.05% and a 95% level of confidence. Thus, 345 goats and sheep were chosen as the overall sample size. However, the sample size was enlarged two times to maximize precision, resulting in 690 goats and sheep being sampled for the seroprevalence investigation (Thrusfield, 2018).

Sampling techniques and sample collection
A multistage sampling technique was carried out to select the zone from the southern region. Among different zones of the region, the zones (Jinka and Wolaita) and districts (Kolme and Abala Abaya districts) were selected purposively based on the population density of sheep and goats. Peasant association: three from Kolme (Masoya, Abaya, and Gete) and three from Abala Abaya (Abala Maraka, Abala Sipa, and Abala Faracho), households, and the herd were selected randomly from both districts. The households that have an average animal number of 10 in Abala Abaya districts and 30 in Kolme districts were included to collect the sample. Accordingly, a total of 690 small ruminants (n = 546 goats and n = 144 sheep) were sampled.
After handling the animal carefully, about 10 ml of blood was obtained aseptically from the jugular vein of each animal using plain vacutainer tubes. Using a permanent marker, all samples were serially identified and correctly labelled. The blood sample was spun at 3000 rpm for 4 min before being gently split into 2 ml cryovials tubes and delivered to the Sodo Regional Veterinary Laboratory using an icebox. The Rose Bengal plate test (RBPT) was used to screen sera samples. After using the RBPT test, all positive samples were sent to the National Veterinary Institute for confirmation using the complement fixation test (CFT) (OIE, 2018).

Rose Bengal plate test
The whole serum sample was first examined using the RBPT, which was

Complement fixation test
The CFT was used to confirm the results of those sera that tested positive by the RBPT. Anti-Brucella antibodies were detected in the serum using standard B. abortus antigen for CFT (from the Veterinary Laboratories Agency, Addlestone, United Kingdom). The control sera and complement were obtained from the German Federal Institute for Consumer and Veterinary Medicine in Berlin. The minimal positive threshold was set at a 25% haemolytic response at a dilution of 1:5.
Sera with a strong reaction of approximately 100% fixation of the complement (4+) at a dilution of 1:5, sera with about 75% fixation of the complement (3+) at a dilution of 1:10, sera with about 50% fixation of the complement (2+) at a dilution of 1:20, and sera with about 25% fixation at a dilution of 1:40 (+) will be classified as positive (Ashenafi et al., 2007).

2.4.3
Questionnaire survey A pre-tested semi-structured questionnaire was given to animal owners to know their knowledge about brucellosis, consuming raw animal products (milk, blood, or meat), handling aborted fetuses and retained placentas by bare hand, reproductive health problems, isolation of diseased animals from the herd, herd size, and history of contact with another herd. As a result, 138 households (90 from Kolme and 48 from Abala Abaya) took part in the research. The overall sample size for a household interview was calculated according to Cochran (Cochran, 1977).

Data analysis
A Microsoft Excel spreadsheet was used to store the information gathered from the questionnaire and laboratory testing.

Overall seroprevalence of small ruminant brucellosis
The seroprevalence of brucellosis as detected using the RBPT and CFT for each district and kebeles was presented in Table 1. The seroprevalence of Brucella infection in Kolme (5.3%) was greater than in Abala Abaya districts (1.0%) and has shown a statistically significant difference (p < 0.05).

Association of risk factors with seroprevalence of small ruminant brucellosis
When using the CFT, goats had a greater seroprevalence of brucellosis (4.0%) than sheep (0.7%). Except for sex, which showed an insignificant association (p > 0.05) with the seroprevalence brucellosis, a statistically significant difference (p < 0.05) was identified with the occurrence of brucellosis among all other risk variables. Females had a greater seroprevalence of brucellosis (4.0%) than males (1.2%). Only 4.4% of small ruminants in the adult age group were found to be seropositive. Furthermore, seroprevalence was found to be greater in herd sizes >30 (4.0%) and parity >4 (7.3%) when compared to their respective categories (Table 2).

Univariable logistic regression analysis of potential risk factors
On univariable logistic regression analysis, associated risk factors such as district, herd size, parity, age, and history of reproductive health problems (abortion, stillbirth, and retained fetal membrane) had a statistically significant effect on seropositivity (p < 0.05), whereas small ruminant species and sex had no statistically significant effect on seropositivity (p > 0.05) ( Table 3).

Multivariable logistic regression analysis of potential risk factors
Variables with a p-value ≤0.25 were all subjected to multivariable logistic regression analysis to construct the likely model (p < 0.05). As a result, multivariable logistic regression analysis employing this model demonstrated a statistically significant association between the prevalence of small ruminant brucellosis with history of abortion, stillbirth, and retained fetal membrane (Table 4).

Assessment of Awareness among Small Ruminants Owners'
The research areas' household educational level revealed that the majority of small ruminant owners interviewed (62.32%) were illiterate ( Figure 2).
All of the respondents (100%) were unaware of the Brucella infection. Around 79.7% of owners used their bare hands to help their animals during parturition which exposed them to the disease. Because of a lack of information about disease transmission, 67.4% of animal owners did not separate sick animals from healthy ones. Approximately 57.2% of respondents' herds had experienced reproductive health problems such as abortion, stillbirth, or retained fetal membrane, and 75.4% of respondents' herds have had previous contact with other herds during feeding and watering time. Note that 93.5% of those polled said that they ate raw animal products regularly. The majority of households in this study were unable to implement any control measures in response to the widespread abortion in small ruminants. Furthermore, they were uninformed about the illnesses' zoonotic potential (Table 5).
The overall seroprevalence of brucellosis in CFT (3.3%) was found to agree with the previous report (Melese, 2016;Sintayehu et al., 2015;Teklue et al., 2013). In contrast, a higher overall seroprevalence was previously described by Wakene et al. (2017)  The seroprevalence of Brucella infection in Kolme and Abala Abaya districts showed a statistically significant difference (p < 0.05) with the occurrence of brucellosis. Similar findings have been previously reported from different parts of the country (Ashenafi et al., 2007;Deddefo et al., 2015;Mengistu, 2007;Tigist et al., 2011;Yibeltal, 2005). The difference in the seroprevalence of Brucella infection could be associated with unrestricted movement of animals for searching pasture and water nearby watering points, especially during the dry season, environmental factors, breed differences, sample size, and the tests used.
A statistically significant difference in the seropositivity of Brucella infection was observed between species. The seroprevalence of Brucella infection in goats (4.03%) was higher than in sheep (0.7%). Similar to this finding, a higher seroprevalence of Brucella infection ranging from 15.4% to 1.9% has been previously reported (Adugna et al., 2013;Ashenafi et al., 2007;Bekele et al., 2011;Mengistu, 2007;Teshome et al., 2018;Wedajo et al., 2015) in goats and sheep. On contrary, Deddefo et al. (2015) in Arsi and East Shoa recorded a higher seroprevalence of brucellosis in goats (4.9%) and sheep (4.4%) with no statistically significant difference. This difference might be due to the differences in flock sizes and the number of goats and sheep kept in the herd. Most breeds of goats are fully susceptible, but the susceptibility of sheep breeds differs widely (Radostitis et al., 2007). The breed-specific difference in the case of goats may be due to persistent infection of mammary glands and supramammary lymph nodes because of constant or intermittent shedding of organisms within the milk during succeeding lactations. However, in the case of sheep, the differences may due to the self-limiting nature of the disease which is seldom accompanied by prolonged excretion of the bacteria (Radostitis et al., 2007).
The seropositivity of Brucella infection between sex was not revealed as a statistically significant difference as previously described by Ashenafi et al. (2007). The lack of statistically significant difference between sexes could be due to the smaller sample size of males, and the habit of keeping males in the herd for a shorter period may decrease their exposure to the disease. Yet, Adugna et al. (2013) have also reported that males are less susceptible to Brucella infection due to the absence of erythritol.
A statistically significant difference that was observed among age groups with the occurrence of brucellosis was found to be in line with Radostits et al. (2006) and Quinn et al. (2004) who have reported that brucellosis is essentially a disease of sexually mature animals. However, young animals may acquire latent infections even though they are more resistant to Brucella infection as they frequently clear an established infection (Walker, 1999). This is due to sex hormones and erythritol, which stimulate the growth and multiplication of Brucella organisms, which tend to increase in concentration with age and sexual maturity (Radostits et al., 2000).
In this study, a statistically significant difference (p < 0.05) was observed between herd-level prevalence and individual animal-level prevalence of brucellosis in the Kolme and Abala Abaya districts. This result was in agreement with the report of Wakene et al. (2017) and Adugna et al. (2013) who appreciated a high seroprevalence of brucellosis at the herd level but contrary to Mohammed et-al. (2017) and Ferede et al. (2011). Although a statistically significant association was observed as parity increased with the seropositivity of brucellosis. This was compatible with Melese (2016), Ashagrie et-al. (2011), andDeddefo et al. (2015). This difference could be due to the effect of reproductive physiology and hormone on the genetic performance of individual animals and differences in the animal population in their respective niche. Furthermore, ewes having a history of abortion, stillbirth, and retained fetal membranes have revealed a statistically significant difference with the seroprevalence of brucellosis. This finding was in agreement with the report of Wakene et al. (Wakene et al., 2017) but contrary to Deddefo et al. (2015) and Adugna et al. (2013).
The difference might be due to the effect of the reproductive and sexual hormones on the pathogenesis of the disease in small ruminants (Radostits et al., 2000).
Among small ruminant owners, 62.32% were illiterate. However, 100% of owners were unaware of the disease, and 79.7% of them used their bare hands during parturition. This was in agreement with the report of Wakene et al. (2017). This study also revealed that 67.4% of owners are not aware of the transmission of brucellosis through contact with other herds during feeding and watering times, and consumption of raw animal products, so they have not practiced the isolation of healthy animals from diseased ones. Approximately 57.2% of respondents' herds had experienced reproductive health problems such as abortion, stillbirth, or retained fetal membrane due to a lack of using any control measure to prevent abortion in small ruminants.
Hence, this is the most common means of exposure of the rural community to the disease in endemic areas as reported by Olsen and Palmer (2014).
Brucellosis is a severe health danger for the entire community due to close contact between humans and their animals, which sometimes share the same housing enclosures. In Ethiopia, reproductive issues such as abortion, stillbirth, and retained fetal membranes are the most common causes of reproductive wastage in small ruminants, and humans may become infected while handling infected material. It has yet to be determined if brucellosis or malaria is the source of human illness with symptoms such as fever, aches and pains, weariness, and debility. Consequently, there is a high risk of human brucellosis in the study area. This outcome is in agreement with the study conducted by Ashenafi et al. (2007) and Habtamu et al. (2015).

CONCLUSION AND RECOMMENDATIONS
This study revealed a high seroprevalence of brucellosis in goats as compared to sheep in Kolme and Abaya Abala districts. All potential risk factors, including district, herd size, parity, age, and a history of reproductive health problems (abortion, stillbirth, and retained fetal membrane), have shown a statistically significant difference with the occurrence of small ruminant brucellosis. In the current study area, smallholder farmers were found to be at high risk of acquiring Brucella infection due to a tradition of ingesting raw milk, traditional animal husbandry practices, handling animals with reproductive difficulties with their bare hands, and lack awareness on the zoonotic nature of the disease. As a result, improved management systems and the implementation of appropriate control measures in the smallholder production system are recommended in the study areas. Further epidemiological research, as well as the identification and isolation of the Brucella biotype responsible for the infection, should be carried out.

AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.

CONFLICTS OF INTEREST
All authors declare conflict of interest.

FUNDING INFORMATION
This work was not supported by any funding source or institution.

DATA AVAILABILITY STATEMENT
The datasets used and analyzed during the current study will be available from the corresponding author on reasonable request.

PEER REVIEW
I would not like my name to appear with my report on Publons https:// publons.com/publon/10.1002/vms3.992.

ETHICS STATEMENT
The Arbaminch University of Research Ethics and Review Committee provided ethical approval for this research. Before collecting blood samples, verbal consent was also sought from owners to take samples from their sheep and goats following strict hygienic measures. The best practice guidelines for animal care were followed as to the purpose of the study, and that the Arbaminch University of Research Ethics and Review Committee approved the verbally informed consent process.